Altered Fermentation Performances, Growth, and Metabolic Footprints Reveal Competition for Nutrients between Yeast Species Inoculated in Synthetic Grape Juice-Like Medium

Abstract

The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance, fermentations performed with K. marxianus were characterized by the formation of phenylethanol and phenylethyl acetate, while those performed with P. burtonii or Z. meyerae displayed higher production of isoamyl alcohol and ethyl esters. When considering sequential inoculation of yeasts, the nutritional requirements of the yeasts used should be carefully considered and adjusted accordingly. Finally, our chemical data suggests that the organoleptic properties of the wine are altered in a species specific manner.

A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae

“…As already observed in a previous study (Rollero et al ., ), the pure culture of S. cerevisiae reached dryness, while fermentation conducted by K. marxianus terminated with 100 g l −1 residual sugars (corresponding to a consumption of 60% of sugar content) (Fig. ).…”

“…Overall, the genes involved in the Ehrlich pathway (see Fig. C) showed a higher level of expression in K. marxianus , providing an explanation for the previously reported higher production of higher alcohols by this species (Rollero et al ., ). However, the expression of the genes responsible for the conversion of α‐ketoisovalerate to α‐ketoisocaproate ( Leu1/2/4/9 ) was lower in K. marxianus .…”

“…Their involvement in fermentation yields distinct and sometimes desirable chemical and aroma profiles different from those produced by S. cerevisiae alone. Kluyveromyces marxianus is a biotechnologically relevant yeast species, and its application in wine fermentation has recently been proposed because of various potentially interesting features as enzymatic activity and aroma production (Rollero et al, 2018a;Rollero et al, 2018b;Rollero et al, 2018c). Existing data suggest that, under winemaking conditions, this yeast displays a similar nitrogen consumption profile to S. cerevisiae, except for ammonium.…”

“…Existing data suggest that, under winemaking conditions, this yeast displays a similar nitrogen consumption profile to S. cerevisiae, except for ammonium. Indeed, K. marxianus consumes only 50% of this compound during wine fermentation while it is depleted by S. cerevisiae (Rollero et al, 2018a). Moreover, K. marxianus also but under fermentative conditions produces higher concentrations of volatile, such as isobutanol, phenylethanol and phenylethyl acetate (Rollero et al, 2018b).…”

Challenges of the Non-Conventional Yeast Wickerhamomyces anomalus in Winemaking

“…Wine fermented with a mixed starter of Pichia burtonii/S. cerevisiae contained higher amounts of ethyl esters [88], whilst co-inoculation of P. kluyveri and S. cerevisiae increased varietal aromas, mainly 3-mercaptohexyl acetate (3MHA) and 3-sulfanylhexan-1-ol in Sauvignon Blanc wines [81,89]. By contrast, a different P. kluyvery isolate did not show a sensorial significant increase in the tropical fruity aromas characterized by 3MHA, and the production of 3-methyl-butanoic acid was associated with an off-putting sour, sweaty, and cheesy aroma that is considered a wine fault [90].…”